1. Field of the Invention
This invention pertains to therapeutic wound healing compositions for protecting and resuscitating mammalian cells (Embodiment One (I)). This invention also pertains to therapeutic antiviral-wound healing compositions for reducing viral titers and increasing the proliferation and resuscitation rate of mammalian cells (Embodiment Two (II)).
In Embodiment One (I), the therapeutic wound healing compositions are used alone. In a first aspect of Embodiment One (I.A), the therapeutic wound healing composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells. In a second aspect of Embodiment One (I.B), the therapeutic wound healing composition comprises (a) pyruvate selected from the group consisting of pyruvic acid, pharmaceutically acceptable salts of pyruvic acid, and mixtures thereof, (b) lactate selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof, and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells. In a third aspect of Embodiment One (I.C), the therapeutic wound healing composition comprises (a) an antioxidant and (b) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells. In a fourth aspect of Embodiment One (I.D), the therapeutic wound healing composition comprises (a) lactate selected from the group consisting of lactic acid, pharmaceutically acceptable salts of lactic acid, and mixtures thereof, (b) an antioxidant, and (c) a mixture of saturated and unsaturated fatty acids wherein the fatty acids are those fatty acids required for the repair of cellular membranes and resuscitation of mammalian cells.
In Embodiment Two (II), the therapeutic wound healing compositions of Embodiment One (I.A-D) are combined with a therapeutically effective amount of an antiviral agent (V) to form antiviral-wound healing compositions (II.A-D+V). This invention also pertains to methods for preparing and using the antiviral-wound healing compositions and the topical and ingestible pharmaceutical products in which the therapeutic compositions may be used.
2. Description of the Background
Wound Healing
Wounds are internal or external bodily injuries or lesions caused by physical means, such as mechanical, chemical viral, bacterial, or thermal means, which disrupt the normal continuity of structures. Such bodily injuries include contusions, wounds in which the skin is unbroken, incisions, wounds in which the skin is broken by a cutting instrument, and lacerations, wounds in which the skin is broken by a dull or blunt instrument. Wounds may be caused by accidents or by surgical procedures. Patients who suffer major wounds could benefit from an enhancement in the wound healing process.
Wound healing consists of a series of processes whereby injured tissue is repaired, specialized tissue is regenerated, and new tissue is reorganized.
Wound healing consists of three major phases: a) an inflammation phase (0-3 days), b) a cellular proliferation phase (3-12 days), and (c) a remodeling phase (3 days-6 months).
During the inflammation phase, platelet aggregation and clotting form a matrix which traps plasma proteins and blood cells to induce the influx of various types of cells. During the cellular proliferation phase, new connective or granulation tissue and blood vessels are formed. During the remodeling phase, granulation tissue is replaced by a network of collagen and elastin fibers leading to the formation of scar tissue.
When cells are injured or killed as a result of a wound, a wound healing step is desirable to resuscitate the injured cells and produce new cells to replace the dead cells. The healing process requires the reversal of cytotoxicity, the suppression of inflammation, and the stimulation of cellular viability and proliferation. Wounds require low levels of oxygen in the initial stages of healing to suppress oxidative damage and higher levels of oxygen in the later stages of healing to promote collagen formation by fibroblasts.
Mammalian cells are continuously exposed to activated oxygen species such as superoxide (O.sub.2), hydrogen peroxide (H.sub.2 O.sub.2), hydroxyl radical (OH.), and singlet oxygen (.sup.1 O.sub.2). In vivo, these reactive oxygen intermediates are generated by cells in response to aerobic metabolism, catabolism of drugs and other xenobiotics, ultraviolet and x-ray radiation, and the respiratory burst of phagocytic cells (such as white blood cells) to kill invading bacteria such as those introduced through wounds. Hydrogen peroxide, for example, is produced during respiration of most living organisms especially by stressed and injured cells.
These active oxygen species can injure cells. An important example of such damage is lipid peroxidation which involves the oxidative degradation of unsaturated lipids. Lipid peroxidation is highly detrimental to membrane structure and function and can cause numerous cytopathological effects. Cells defend against lipid peroxidation by producing radical scavengers such as superoxide dismutase, catalase, and peroxidase. Injured cells have a decreased ability to produce radical scavengers. Excess hydrogen peroxide can react with DNA to cause backbone breakage, produce mutations, and alter and liberate bases. Hydrogen peroxide can also react with pyrimidines to open the 5,6-double bond, which reaction inhibits the ability of pyrimidines to hydrogen bond to complementary bases, Hallaender et al. (1971). Such oxidative biochemical injury can result in the loss of cellular membrane integrity, reduced enzyme activity, changes in transport kinetics, changes in membrane lipid content, and leakage of potassium ions, amino acids, and other cellular material.
Antioxidants have been shown to inhibit damage associated with active oxygen species. For example, pyruvate and other .alpha.-ketoacids have been reported to react rapidly and stoichiometrically with hydrogen peroxide to protect cells from cytolytic effects, O'Donnell-Tormey et al., J. Exp. Med., 165, pp. 500-514 (1987).
U.S. Pat. Nos. 3,920,835, 3,984,556, and 3,988,470, all issued to Van Scott et al., disclose methods for treating acne, dandruff, and palmar keratosis, respectively, which consist of applying to the affected area a topical composition comprising from about 1% to about 20% of a lower aliphatic compound containing from two to six carbon atoms selected from the group consisting of .alpha.-hydroxyacids, .alpha.-ketoacids and esters thereof, and 3-hydroxybutryic acid in a pharmaceutically acceptable carrier. The aliphatic compounds include pyruvic acid and lactic acid.
U.S. Pat. Nos. 4,105,783 and 4,197,316, both issued to Yu et al., disclose a method and composition, respectively, for treating dry skin which consists of applying to the affected area a topical composition comprising from about 1% to about 20% of a compound selected from the group consisting of amides and ammonium salts of .alpha.-hydroxyacids, .beta.-hydroxyacids, and .alpha.-ketoacids in a pharmaceutically acceptable carrier. The compounds include the amides and ammonium salts of pyruvic acid and lactic acid.
U.S. Pat. No. 4,234,599, issued to Van Scott et al., discloses a method for treating actinic and nonactinic skin keratoses which consists of applying to the affected area a topical composition comprising an effective amount of a compound selected from the group consisting of .alpha.-hydroxyacids, .beta.-hydroxyacids, and .alpha.-ketoacids in a pharmaceutically acceptable carrier. The acidic compounds include pyruvic acid and lactic acid.
U.S. Pat. No. 4,294,852, issued to Wildnauer et al., discloses a composition for treating skin which comprises the .alpha.-hydroxyacids, .beta.-hydroxyacids, and .alpha.-ketoacids disclosed above by Van Scott et al. in combination with C.sub.3 -C.sub.8 aliphatic alcohols.
U.S. Pat. No. 4,663,166, issued to Veech, discloses an electrolyte solution which comprises a mixture of L-lactate and pyruvate in a ratio from 20:1 to 1:1, respectively, or a mixture of D-.beta.-hydroxybutyrate and acetoacetate, in a ratio from 6:1 to 0.5:1, respectively.
Sodium pyruvate has been reported to reduce the number of erosions, ulcers, and hemorrhages on the gastric mucosa in guinea pigs and rats caused by acetylsalicylic acid. The analgesic and antipyretic properties of acetylsalicylic acid were not impaired by sodium pyruvate, Puschmann, Arzneimittelforschung, 33, pp. 410-415 and 415-416 (1983).
Pyruvate has been reported to exert a positive inotropic effect in stunned myocardum, which is a prolonged ventricular dysfunction following brief periods of coronary artery occlusions which does not produce irreversible damage, Mentzer et al., Ann. Surg., 209, pp. 629-633 (1989).
Pyruvate has been reported to produce a relative stabilization of left ventricular pressure and work parameter and to reduce the size of infarctions. Pyruvate improves resumption of spontaneous beating of the heart and restoration of normal rates and pressure development, Bunger et al., J. Mol. Cell. Cardiol., 18, pp. 423-438 (1986), Mochizuki et al., J. Physiol. (Paris), 76, pp. 805-812 (1980), Regitz et al., Cardiovasc. Res., 15, pp.652-658 (1981), Giannelli et al., Ann. Thorac. Surg., 21, pp. 386-396 (1976).
Sodium pyruvate has been reported to act as an antagonist to cyanide intoxication (presumably through the formation of a cyanohydrin) and to protect against the lethal effects of sodium sulfide and to retard the onset and development of functional, morphological, and biochemical measures of acrylamide neuropathy of axons, Schwartz et al., Toxicol. Appl. Pharmacol., 50, pp. 437-442 (1979), Sabri etal., Brain Res., 483, pp. 1-11 (1989).
A chemotherapeutic cure of advanced L1210 leukemia has been reported using sodium pyruvate to restore abnormally deformed red blood cells to normal. The deformed red blood cells prevented adequate drug delivery to tumor cells, Cohen, Cancer Chemother. Pharmacol., 5, pp. 175-179 (1981).
Primary cultures of heterotopic tracheal transplant exposed in vivo to 7,12-dimethyl-benz(a)anthracene were reported to be successfully maintained in enrichment medium supplemented with sodium pyruvate along with cultures of interleukin-2 stimulated peripheral blood lymphocytes, and plasmacytomas and hybridomas, pig embryos, and human blastocysts, Shacter, J. Immunol. Methods, 99, pp. 259-270 (1987), Marchoketal., Cancer Res., 37, pp. 1811-1821 (1977), Davis, J. Reprod. Fertil. Suppl., 33, pp. 115-124 (1985), Okamoto etal., No To Shinkei, 38, pp. 593-98 (1986), Cohen et al., J. In Vitro Fert. Embryo Transfer, 2, pp. 59-64 (1985).
U.S. Pat. Nos. 4,158,057, 4,351,835, 4,415,576, and 4,645,764, all issued to Stanko, disclose methods for preventing the accumulation of fat in the liver of a mammal due to the ingestion of alcohol, for controlling weight in a mammal, for inhibiting body fat while increasing protein concentration in a mammal, and for controlling the deposition of body fat in a living being, respectively. The methods comprise administering to the mammal a therapeutic mixture of pyruvate and dihydroxyacetone, and optionally riboflavin. U.S. Pat. No. 4,548,937, issued to Stanko, discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of pyruvate, and optionally riboflavin. U.S. Pat. No. 4,812,479, issued to Stanko, discloses a method for controlling the weight gain of a mammal which comprises administering to the mammal a therapeutically effective amount of dihydroxyacetone, and optionally riboflavin and pyruvate.
Rats fed a calcium-oxalate lithogenic diet including sodium pyruvate were reported to develop fewer urinary calculi (stones) than control rats not given sodium pyruvate, Ogawa et al., Hinyokika Kiyo, 32, pp. 1341-1347 (1986).
U.S. Pat. No. 4,521,375, issued to Houlsby, discloses a method for sterilizing surfaces which come into contact with living tissue. The method comprises sterilizing the surface with aqueous hydrogen peroxide and then neutralizing the surface with pyruvic acid.
U.S. Pat. No. 4,416,982, issued to Tauda et al., discloses a method for decomposing hydrogen peroxide by reacting the hydrogen peroxide with a phenol or aniline derivative in the presence of peroxidase.
U.S. Pat. No. 4,696,917, issued to Lindstrom et al., discloses an eye irrigation solution which comprises Eagle's Minimum Essential Medium with Earle's salts, chondroitin sulfate, a buffer solution, 2-mercaptoethanol, and a pyruvate. The irrigation solution may optionally contain ascorbic acid and .alpha.-tocopherol. U.S. Pat. No. 4,725,586, issued to Lindstrom et al., discloses an irrigation solution which comprises a balanced salt solution, chondroitin sulfate, a buffer solution, 2-mercaptoethanol, sodium bicarbonate or dextrose, a pyruvate, a sodium phosphate buffer system, and cystine.
The irrigation solution may optionally contain ascorbic acid and gamma-tocopherol.
U.S. Pat. No. 3,887,702 issued to Baldwin, discloses a composition for treating fingernails and toenails which consists essentially of soybean oil or sunflower oil in combination with Vitamin E.
U.S. Pat. No. 4,847,069, issued to Bissett et al., discloses a photoprotective composition comprising (a) a sorbohydroxamic acid, (b) an anti-inflammatory agent selected from steroidal anti-inflammatory agents and a natural anti-inflammatory agent, and (c) a topical carrier. Fatty acids may be present as an emollient. U.S. Pat. No. 4,847,071, issued to Bissett et al., discloses a photoprotective composition comprising (a) a tocopherol or tocopherol ester radical scavenger, (b) an anti-inflammatory agent selected from steroidal anti-inflammatory agents and a natural anti-inflammatory agent, and (c) a topical carrier. U.S. Pat. No. 4,847,072, issued to Bissett et al., discloses a topical composition comprising not more than 25% tocopherol sorbate in a topical carrier.
U.S. Pat. No. 4,533,637, issued to Yamane et al., discloses a culture medium which comprises a carbon source, a nucleic acid source precursor, amino acids, vitamins, minerals, a lipophilic nutrient, and serum albumin, and cyclodextrins. The lipophilic substances include unsaturated fatty acids and lipophilic vitamins such as Vitamin A, D, and E. Ascorbic acid may also be present.
United Kingdom patent application no. 2,196,348A, to Kovar et al., discloses a synthetic culture medium which comprises inorganic salts, monosaccharides, amino acids, vitamins, buffering agents, and optionally sodium pyruvate adding magnesium hydroxide or magnesium oxide to the emulsion. The oil phase may include chicken fat.
U.S. Pat. No. 4,284,630, issued to Yu et al., discloses a method for stabilizing a water-in-oil emulsion which comprises adding magnesium hydroxide or magnesium oxide to the emulsion. The oil phase may include chicken fat.
PREPARATION.TM. has been reported to increase the rate of wound healing in artificially created rectal ulcers. The active ingredients in PREPARATION H.TM. are skin respiratory factor and shark liver oil, Subramanyam et al., Digestive Diseases and Sciences, 29, pp. 829-832 (1984).
The addition of sodium pyruvate to bacterial and yeast systems has been reported to inhibit hydrogen peroxide production, enhance growth, and protect the systems against the toxicity of reactive oxygen intermediates. The unsaturated fatty acids and saturated fatty acids contained within chicken fat enhanced membrane repair and reduced cytotoxicity. The antioxidants glutathione and thioglycollate reduced the injury induced by oxygen radical species, Martin, Ph.D. thesis, (1987-89).
U.S. Pat. No. 4,615,697, issued to Robinson, discloses a controlled release treatment composition comprising a treating agent and a bioadhesive agent comprising a water-swellable but water-insoluble, fibrous crosslinked carboxy-functional polymer.
European patent application no. 0410696A1, to Kellaway et al., discloses a mucoadhesive delivery system comprising a treating agent and a polyacrylic acid cross-linked with from about 1% to about 20% by weight of a polyhydroxy compound such as a sugar, cyclitol, or lower polyhydric alcohol.
Viral Infections
Herpes simplex virus type 1 (HSV-1) is a common viral infection in humans which commonly causes epidermal lesions in and around the oral cavity. The hallmark of an HSV infection is the ability of the virus to establish a latent infection in the nervous system, and to reactivate and cause recrudescent lesions. Recurrent disease can be a rather unsightly, painful, and unpleasant episode, Overall J. C. Dermatologic viral diseases; In: Galasso GJ. Merigan TC. Buchanan RA. eds. Antiviral agents and viral diseases on man. 2nd ed. New York: Raven Press. 1984:247-312.
The vast majority of perioral infections are caused by HSV type I and serologic studies indicate that 50% to 100% of the population has contacted the virus by adulthood, Nahmias A. J., Roizman B., Infection with herpes-simplex virus 1 and 2, N. Engl. J. Med. 1973:289:781-789. More important, it is estimated that 20% to 45% of the population have reoccurring perioral HSV infections, most commonly in the form of fever blisters, Young S. K., 1 Rowe N H, Buchanan R. A., A clinical study for the control of facial mucocutaneous herpes virus infections, I.Characterization of natural history in a professional school population, Oral Surg. Oral Med. Oral Pathol., 1976:41:498-507; Embil J. A., Stephens R. G., Manuel F. R., Prevalence of recurrent herpes labialis and aphthous ulcers among young adults on six continents, Can. Med.Assoc. J., 1975: 113:627-30; Ship I. I., Brightman V. J., Laster L. L., The patient with recurrent aphthous ulcers and the patient with recurrent herpes labialis: a study of two population samples, J. Am. Dent. Assoc. 1967:75:645-54. Fever blisters are more than a minor annoyance; an estimated 98 million cases occur each year in the United States, Spruance S. L., Overall J. C. Jr, Kern E. R. et al., The natural history of recurrent herpes simplex labialis: implications for anti-viral therapy, N. Eng. J. Med. 1977:197:69-75. Fever blisters cause considerable discomfort and are esthetically annoying to patients.
The Herpes group of viruses is composed of seven human viruses and multiple animal viruses. The human herpes viruses consist of herpes simplex virus type I and II, varicella-zoster, cytomegalovirus, Epstein-barr, and human herpes virus types 6 and 7. The viruses are similar in size and morphology, and are characterized by a double-stranded DNA core and a lipoprotein envelope with glycoprotein projections. All the human herpes viruses replicate primarily in the cell nuclei. HSV-I and HSV-2 can be distinguished by a variety of properties, including clinical and epidemiologic patterns, antigenicity, DNA base composition, biologic characteristics, and sensitivity to various physical and chemical stresses, Rodu B., Russell C., Mattingly G., Determining therapeutic efficacy in recurrent herpes labialis by lesion size analysis, Oral Surg. Oral Med. Oral Pathol. Aug 1991:178-182; Fox J. D., Briggs M., et al: Human herpes virus 6 in salivary glands, Lanced 336:590. 1990; Cory L., Spear P. G.: Infection with herpes simplex viruses (pts 1 and 2). N. Engl. J. of Med. 314:686,749, 1986; Hammer S. M., et al.: Temporal cluster of herpes simplex encephalitis: investigation by restriction endonuclease cleavage of viral DNA, J. Infect. Dis. 141:436, 1980; Johnson R. E., Nahmias A. J., et al.: A seroepidemiologic survey of the prevalence of herpes simplex virus type 2 infection in the United States, N. Engl. J. Med. 321:7, 1989.
HSV-I primary infections occur mainly in childhood. The herpes virus is a contact infectious agent that invades the moist membranes of the lips, mouth, or throat. The herpes virus is most frequently transmitted by kissing. Although virus titers are higher and transmission is more likely when lesions are present, a symptomatic excretion of the virus is common. Thus, the virus may be transmitted even in the absence of lesions.
On entry into the skin sites, the virus replicates in epithelial cells, which results in lysis of infected cells and the instigation of a local inflammatory response. After primary infection, the virus may become latent within sensory nerve ganglion sites, Bonneau R. H., Sheridan J. F., et al. Stress-induction suppression of herpes simplex virus (HSV)-specific cytotoxic T lymphocyte and natural killer cell activity and enhancement of acute pathogenesis following local HSV infection, Brain, Behavior and Immunity 5, 170192, 1991; Rooney J. F., et al. Prevention of ultraviolet-light-induced herpes labialis by suncreen. The Lancet: 338:1419-1422.1991; Bastian F. O., Rabson A. S., Yee CL, et al., Herpes virus hominis:isolation form human trigeminal ganglion, Science, 1972: 178:306. In humans, the virus remains in a dormant state in the trigeminal ganglion near the cheek bone. The virus can remain dormant in these nerve cells for extended periods of time. The virus can emerge from latency, track along the neural pathway back to the site of the original infection causing the formation of a cold sore blister. A variety of humoral and cell-mediated immune mechanisms are recruited in response to primary and recurrent HSV infections including the production of antibodies, interferon, activation of macrophages, the induction of T-Lymphocyte-mediated reactivity, and the development of antibody dependent lymphocyte cytotoxicity, Bonneau R. H., Sheridan JF, et al., Stress-induction suppression of herpes simplex virus (HSV)-specific cytotoxic T lymphocyte and natural killer cell activity and enhancement of acute pathogenesis following local HSV infection, Brain, Behavior and Immunity 5, 170192, 1991.
Once infected, the cold sore manifests itself in the form of a fluid filled blister inside or outside the mouth. Other possible symptoms which occur three to five days after exposure to the virus include: fever, swollen neck glands, and general aches and pain. As time passes, the fever blister will collapse followed by the formation of yellow crust over the sore. The entire sore usually heals without scaring within two weeks, Bonneau R. H., Sheridan J. F., et al. Stress-induction suppression of herpes simplex virus (HSV)-specific cytotoxic T lymphocyte and natural killer cell activity and enhancement of acute pathogenesis following local HSV infection, Brain, Behavior and Immunity 5, 170192, 1991; Rooney J. F., et al. Prevention of ultraviolet-light-induced herpes labialis by suncreen. The Lancet: 338:1419-1422.1991.
Recurrent infections are generally less severe than the primary attack. Recurrent infections decline in frequency after the age of 35. Signals of recurrent infections include: itching, tingling, burning in the lip area, one to three days before the blister forms. In the United States, lip or perioral recurrences develop in 20 to 40% of the population. Recurrences vary in frequency from more than one attack per month to less than one attack every six months. Factors triggering recurrence are: emotional stress, fever, illness, injury, overexposure to the sun, and menses. Sunlight triggers herpes labialis in approximately 25% of people with recurrent infections.
The key to prevention is to eliminate exposure to the virus. When cold sores are present, one can prevent against autoinoculation by not touching the sores. One can prevent against spreading the virus by not kissing other individuals. Currently cold sores cannot be cured. Treatment exists for relief of pain and discomfort. Antiseptic creams, emollients, and antiseptic ingredients reduce the discomfort through their cooling and protective actions. Sun blocks with UVB protection act as a prophylaxis for people prone to recurrent cold sore sun blisters.
Several drugs are currently available for the treatment of HSV infections. Acyclovir (trade name Zovirax) is a prescription compound that interferes with viral DNA replication through its action on viral thymidine kinase. Although extremely effective when given orally or intravenously for the treatment of primary or encephalitic HSV infections, Acyclovir has little effectiveness, and is not generally prescribed, for recurrent disease. A variety of over-the-counter medications are also available. Most of these medications rely on the weak antiviral properties of chemicals such as phenol which has a low level of effectiveness against recurrent HSV infections, Whitley R. J., Gnann J. W., Acyclovir a decade later, N. Eng. J. of Med. September 1992,782-89.
While the above therapeutic wound healing compositions are reported to inhibit the production of reactive oxygen intermediates, none of the above compositions are entirely satisfactory. None of the compositions has the ability to simultaneously decrease cellular levels of hydrogen peroxide production, increase cellular resistance to cytotoxic agents, increase rates of cellular proliferation, increase cellular viability to protect and resuscitate mammalian cells, and reduce virus titers. The present invention provides such improved therapeutic antiviral-wound healing compositions without the disadvantages characteristic of previously known compositions. This invention also relates to methods for preparing and using the therapeutic antiviral-wound healing compositions and the topical and ingestible pharmaceutical products in which the therapeutic compositions may be used.